Accurately detecting light in a capillary electrophoresis system is an increasingly vital procedure for the analysis of chemicals, cells and biological matter.
This invention relates to a capillary electrophoresis system, particularly where light through a capillary tube is optically detected. More specifically, the invention relates to the precise detection of light output from such a capillary electrophoresis system. Additionally, the invention is concerned with optimizing light energy input to and output from a capillary electrophoresis tube so that, overall, there is obtained a highly sensitive capillary electrophoresis system.
Electrophoresis is an analytical technique whereby small volumes of mixed sample solution are separated by differences in electric charges and molecular sizes of individual sample components. Capillary electrophoresis requires the transportation of small, often minute, quantities of sample solution through a capillary tube under pressure or electrical differential. As the sample travels though the capillary tube, a separation of components of the sample is effected due to the differential.
A light source and a light detector are placed outside the capillary tube which is mounted in a support. As the sample, so separated, migrates through the capillary tube, light is passed along an optical path across the sample. By detecting the light output, information about the nature, for instance, the chemical make-up, of the sample can be obtained.
The capillary electrophoresis tube is a microbore tube and is used as the support and means for transporting liquid containing the sample. Typically, the dimension of the capillary bore diameter ranges from 5 to 500 microns. The shape usually employed for the capillary tube is cylindrical and the wall thicknesses of the tube ranges from 25 to 200 microns.
The nature of the walls of the capillary tube provide different refraction indices and generally cause inaccuracies in the light which is received by the detector. To provide accurate results, however, it is important to avoid optical problems such as distortions to the light caused by perturbations and wall effects of the tube.
The small sizes encountered in the capillary dimensions pose severe problems for accurate optical detection. These include problems arising from the short pathlength through the sample.
Detection of the separated components requires a measurable property of the component. Absorbance, for instance, is measured as the relative decrease in the intensity of the light at a selected wavelength passing through the sample due to the relative concentration of the sample being measured, its specific absorbtivity, and pathlength.
Absorbance is expressed by the Beer-Lambert-Bourgier Law: EQU A=kcl
where
A=Absorbance PA1 k=molar extinction coefficient PA1 c=concentration of the absorbing sample PA1 l=pathlength through sample
When the pathlength becomes smaller, as is encountered with the minute capillary dimensions typically used, the magnitude of the Absorbance decreases.
With the circular cross section of the bore of the capillary tube, the pathlength also varies sinusoidally as light passes transversely through the capillary bore. The pathlength at the poles is zero, and is a maximum length at the equator.
Another problem occurs when light from the light source passes through the solid wall material of the capillary tube without passing through the sample solution in the bore of the tube. This light, termed stray light, contributes to the light energy arriving at the detector without having been attenuated by the absorbing sample.
Additional problems arise in the optical system for detection of solution volumes at the tiny dimensions used for capillaries. To assure reproducible reliable determinations of detected light, the capillary tube must be positioned in the optical system rigidly and accurately. It is difficult and costly to achieve the mechanical tolerances required to meet these conditions where the bore of the tube is movable relative to the window for light that enters the bore.
There is a need to provide a system which can provide accurate data and information within modern detail standards and yet have relaxed precision mechanical tolerances and requirements.
The prior art has used optical systems and optical devices such as lenses and slits in the optical path to improve the optical system. These devices themselves create optical distortions and changes, such as dispersion, to the light. Further inaccuracies are thus created in the detected light.
There is a need to provide a system with a minimum number of optical elements and devices between the signal input and the detected signal.
Light for the optical system is obtained and received through fiber optical input and output. The fiber optic has a core and cladding which have different refractive indices, and the light is propagated in the fiber core. An Angle of Acceptance of the fiber is the half-angle of an Acceptance cone of the fiber. This is the angle about the central axis of the fiber. It is also the angle at the interface of the core and cladding, namely, the Angle of Acceptance is defined by the difference in the refractive index of the core and cladding. Light entering a fiber at angles greater than the acceptance angle leak away and are not propagated to the output end of the fiber optic. Similarly, light normally does not exit a fiber at an angle greater than the Angle of Acceptance.
A Numerical Aperture of the fiber is related to the Angle of Acceptance through the fiber, and is a measure of light-gathering power of the fiber optic. The Numerical Aperture is the sine of the Angle of Acceptance for the fiber.
Prior art systems have not been able to optimize the optical system and the relationship of light generation and propagation in a fiber optic and the detection characteristics in capillary electrophoresis systems.
There is a need for an improved capillary electrophoresis detection system having less distortion of light, and which is easily configured with the fiber optics of the optical system. Also, there is a need for a system which optimizes the use of the light energy from a fiber optic as measured by the Numerical Aperture into the capillary electrophoresis tube. Further, there is in turn, the need for the output from the capillary electrophoresis tube to be related to a receiving fiber optic so as to maximize the receipt of the light from the tube as measured by the Numerical Aperture of the receiving fiber optic.